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Original Article Comparison of Salivary Cotinine Concentration in Cigarette Smokers, Water Pipe Smokers and Non Smokers M. Rabiei 1, M. Rahbar Taramsar 2, M. Sadegh Kanjani 3, E. Kazemnezhad Leyli 4, H. Masoudi Rad 5, M. Jafari Farshami 6. 1 Associate Professor, Department of Oral and Maxillofacial Medicine, School of Dentistry, Guilan University of Medical Sciences. Guilan, Iran 2 Assistant Professor, Department of Legal Medicine and Toxicology, School of Medicine, Guilan University of Medical Sciences. Guilan, Iran 3 PHD Reference laboratory, Guilan University of Medical Sciences. Guilan, Iran 4 Assistant Professor, Department of Biostatistics, School of Medicine, Guilan University of Medical Sciences. Guilan, Iran 5 Assistant Professor, Department of Endodontics, School of Dentistry, Guilan University of Medical Sciences. Guilan, Iran 6 Dentist Corresponding author: M. Rabiei, Associate Professor, Department of Oral and Maxillofacial Medicine, School of Dentistry, Guilan University of Medical Sciences. Guilan, Iran Received: 28 Jan 2013 Accepted: 17 August 2013 Abstract Background and Aim: Water pipe smoking has become a public health hazard. The aim of this study was to measure and compare the concentration of salivary cotinine in cigarette smokers, water pipe smokers and nonsmokers. Materials and Methods: Forty-eight volunteers (16 cigarette smokers, 16 water pipe smokers and 16 nonsmokers) participated in this cross-sectional study. High sensitivity Salivary Cotinine Quantitative enzyme immunoassay kit was used to measure the salivary cotinine concentration. Non-parametric Kruskal Wallis test, Pearson s correlation coefficient and linear regression were used to determine the correlation between number of cigarettes smoked and concentration of cotinine. Results: The mean concentration of salivary cotinine was (±181.56) ng/ml (the highest) in cigarette smokers, (±135.23) ng/ml in water pipe smokers and 0.73(±1.24) ng/ml in nonsmokers. The difference in this respect among the 3 groups was statistically significant (P 0.01). In smokers, the level of salivary cotinine increased by 1.84 ng/ml per each time of cigarette smoking per week. This increase was ng/ml per each time of water pipe consumption per week. Conclusion: The mean concentration of salivary cotinine was significantly higher among cigarette smokers compared to water pipe smokers and nonsmokers. However, one time consumption of water pipe caused a greater rise in salivary cotinine level compared to cigarette smoking. Key Words: Cotinine, Saliva, Smoking, Tobacco The Journal of Islamic Dental Association of IRAN (JIDA) Winter 2014 ;25, (4) Introduction Hookah was invented in the 15 th century in India and quickly became popular in the Middle Eastern countries likeiran, Syria, Yemen, and Egypt [1]. It underwent a transformation from its rudimentary coconut-and-straw look to its current form, a glass vessel with hoses attached to the body [2,3]. In 1990, Arabic countries added floral and fruity flavors to tobacco and made it milder called Maasel derived from the word Mua sel meaning honey [2-3]. At present, hookah smoking is popular worldwide mostly attributed to misconceptions about its safety.its popularity among the youthis due toits social acceptance, novel design, availability in numerous appealing flavors, and low cost [4]. Jan 2014; Vol. 25, No The Journal of Islamic Dental Association of IRAN (JIDA) Winter 2014 ;25, (4) Rabiei et. al In a study conducted in Bandar Abbas, Iran, the prevalence of smoking was 11.7% and mainly in males; whereas 9.1% mentioned hookah smoking including 6.8% of females and 2.3% of males [5]. Hookah smoking is not considered as bad ascigarette smoking in many countries and young adults in the age of 5-18 yrs. are even invited to smoke hookah by their family members [4]. Hookah smoking is becoming popular in developed countries as well. Unlike cigarette smoking,the American Food and Drug Administration (FDA) has no supervision over hookah tobacco packaging and only a no tar label isdisplayed on hookah tobacco packs [4]. It is estimated that smoking will cause 10 million deaths annually by the year This rate will be much higher in developing countries compared to developed ones. Moreover, it is predicted that by the year 2030, 70% of annual deaths from smoking worldwide will occur in developing countries [5]. Tobacco leaf combustion is an incomplete process producing a gaseous phase consisting of carbon monoxide, nitrosamine, acetaldehyde, formaldehyde, volatile hydrocarbons, and hydrogen cyanide and particulate matter phase that is essentially unfiltered consisting of tar and nicotine. The gaseous phase contains more carcinogens [6,7]. Cigarette smoke contains free radicals that can cause tissue damage by reacting with unsaturated fatty acids in cell membranes and DNA nucleotides [8]. Studies have shown that smoking hookah significantly increases the prevalence of many diseases, such as lung cancer, lung disease, weight loss and periodontal disease. In some cases this increase has not been significant, but an increased risk of bladder cancer, esophageal cancer, oral dysplasia and infertility as the result of hookah smoking has been noticed [9]. Cotinine is an alkaloid found in tobacco and also a nicotine metabolite. Cotinine is also an anagram of nicotine andis used as a biomarker for measurement ofexposure to tobacco smoke [10]. Also, due to its relatively long half-life ofapproximately 20 hours(compared to2 hours for nicotine) in body fluids, it has optimal sensitivity and specificity for measuring tobacco exposure instead of nicotine [11]. Cotinine serum level of 10 ng/ml is considered as the breakpoint between smokers and nonsmokers. This rate is 2 ng/ml and 5 ng/mlin urine and saliva, respectively [12,13]. At present, saliva is considered a reliable alternative to other body fluids for measurement of cotinine concentration. Measurement of salivary cotinine is affordable, convenient and non-invasive requiring no expertise in sample collection.the correlation between salivary and serum cotinine levels has been confiremd.thus, saliva seems to be an ideal alternative to serum for this purpose [14,15]. This study sought to compare the mean level of salivary cotinine in hookah smokers, cigarette smokers and nonsmokers. Materialsand Methods This comparative cross-sectional study was conducted in Rasht,Iran in The study design was approved in the Ethics Committee ofgilanuniversity. After obtaining written informed consent, 48 volunteers, divided into 3 groups of cigarette smokers, hookah smokers and nonsmokers were evaluated. The inclusion criteria were age between 18 to 25 yrs. and Body Mass Index (BMI)of18.5 to 25 (normal range).all participants were males and matched in terms of age (3 ± yrs.). The cigarette used by cigarette smokers was Winston Light with anicotine level of 0.6 mg per cigarette. The tobacco used by hookah smokers was Nakhla Tobacco with 0.5%/g nicotine level. Medium size waterpipes were used in thisstudy. The type and amount of charcoalswere also the same. The experiment was done in an open-air environment in order to eliminate the effect of secondhand smoke on participants. The smoker groups were requested not to use any other tobacco products during the course of study or they will be excluded from the study. Before the experiment, participants were thoroughly informed about the process of study and matching the conditions among hookah smokers. The experiment was carried out in an outdoor cafe in the city of Rasht. The number of tobacco consumptionsper week was also recorded. Participants smoked hookah 20 hours prior to salivary sampling. For eachhookah smoking, 20 grams of tobacco was used containing 1 mg of nicotine. The mean time of smoking wasapproximately 45 minutes and during this time period smokers had different numbers of puffs. Participants were 268 Jan 2014; Vol. 25, No. 4 Rabiei et. al Comparison of Salivary Cotinine Concentration in Cigarette... asked to refrain from smoking or exposure to smoke for the next 20h until the salivary sampling. Cigarette smokers smoked a cigarette at the same place under the same conditions. Smokers were asked to refrain from smoking or exposure to smoke for the next 20h until the salivary sampling. At the time of sampling, subjects were asked againif they had smokedor beenexposed to smoke in the past 20 hours (half-life of salivary cotinine based on the Salimetrics cotinine kit manual is 17 hours). This was done to exclude those who answered positively. Before sampling, hookah and cigarette smokers were asked to write down the average number of Hookah smoking sessions and cigarettes smoked per week, respectively. Salivary samples were then obtained from the two smoker groups and thenonsmokers. Salivary samples were collected by spitting. Volunteers were first asked to wash their mouths, hold their saliva for at least 5 minutes and then spit into a test tube and funnel glass. All subjects had a minimum of 5 ml of salivary samples taken. All salivary samples were collected between 11 am to 1 pm, and sent to the Reference Laboratory of Rasht by cold chain. In the laboratory, using a 10µL pipette, each salivary samplewas divided into at least 2 Eppendorf tubes of 1 ml volume. The tubes containing salivary samples were then frozen in an ultra-low temperature freezer at -75 o C. Salivary cotinine analysis was conducted by using a Sailmetrics (USA, PA) high sensitivity salivary cotinine quantitative enzyme immunoassay kit and enzyme-linked immunosorbent assay (ELISA). First, all salivary samples were removed from the freezer and kept at room temperaturefor 30 minutes (for liquefaction). Samples were centrifuged at 30 rpm for 15 minutes using a TadghizGostar microcentrifuger. Samples were the preparedfollowing the kit instructions. The normality of cotinine distribution was studied using One Sample Kolmogorov-Smirnov test. Considering the normality of cotinine changes in smoker groups, the independent t-test was applied to compare the mean cotinine changes. Due to the lack of normality of cotinine distribution, the mean changes of cotinine between cigarette smokers and nonsmokers and also hookah smokers and nonsmokers were compared using the Mann-Whitney test. Due to the lack of normality, nonparametric Kruskal-Wallis test was applied to compare salivary cotinine levels among the 3 groups. Pearson s correlation coefficient was used to assess the relationship between salivary cotinine level, duration and frequency of cigarette smoking and hookah consumption. IBM SPSS version16 software was used for statistical analysiswith a significance level of p 0.05. Results In this study, 48 subjects were divided into three groups of 16 hookah smokers, 16 cigarette smokers and16 nonsmokers. Each group was evaluated in terms of the salivary cotinine level. The mean age was 23.50±1.26 years, 24.18±0.91 years and ±1.2 years in hookah smokers, cigarette smokers and nonsmokers, respectively. Results showed that the mean and standard deviation of cotinine was ± ng/mlin hookah smokers, ± ng/ml in cigarette smokers and 0.73±1.24 in the control group with 95% CI. Comparison of cotinine levels among the 3 groups using the nonparametric Kruskal-Wallis test revealedsignificant differences among the three groups (p 0.01). The mean cotinine level was significantly different between the cigarette and hookah smokers and the nonsmoker group (p 0.01). Independent t-test demonstrated a significantly higher cotinine level in cigarette smokers compared to hookah smokers (p 0.04) (Table 1). Cigarette smokers averagely smoked (±92.93) cigarettes per week while hookah smokers smoked hookah averagely (±15.79) times a week. A correlation existed between the salivary cotinine level and frequency of smoking in both smoking groups (P 0.01, r=0.943 for cigarette smokers and P 0.033, r=0.535 for hookah smokers both statistically significant). Table 2 shows the regression coefficients of the effect of tobacco consumption on level of salivary cotininein hookah smokers and cigarette smokers. Linear regression model showed the significant effect of the frequency of tobacco consumption onsalivary cotinine level in hookah smokers and cigarette smokers (P 0.033 for hookah and P 0.01 for cigarette smokers). In cigarette smokers, smoking one cigarette per week increased the cotinine level by averagely 1.84 ng/ml (95% Jan 2014; Vol. 25, No The Journal of Islamic Dental Association of IRAN (JIDA) Winter 2014 ;25, (4) Table 1: Level of salivary cotinine in the 3 groups with 95% CI Rabiei et. al Variables Number Mean Standard deviation Minimum Maximum Hookah smokers Cigarette smokers Nonsmokers Total Variable Hookah smokers Cigarette smokers Number of cigarettes smoked ? regression coefficient /062 2/237 0/73 1/102 CI; 1.46, 2.22) while one time hookahsmoking per week increased salivary cotinine by averagely 4.57 ng/ml (95% CI; 0.428, 8.72). Figures 1 and 2 show the linear regression model of correlation between salivary cotinine level and frequency of smoking in hookah and cigarette smokers. Standard error /23 181/56 1/24 157/58 P value Table 2: Regression Coefficients of the effect of smoking frequency on salivary cotinine levels in cigarette and hookah smokers Cotinine level (ng/ml) *Effect of frequency of tobacco use on the salivary cotinine level **Effect of confounding factors (fixed amount) Diagram 1: Linear regression model of the correlation of cotinine with frequency of hookah smoking Cotinine level (ng/ml) 4/ 3/ 2/ / Number of hookah smoking 1/ 2/ 3/ 4/ 5/ sessions 6/ 4/ 2/ 1/ 2/ 3/ 4/ Number of cigarette smoked in a week Diagram 2: Linear regression model of the correlation of cotinine with frequency of cigarette smoking 0. 95% confidence interval for? regression coefficient Minimum Maximum Discussion The highest level of salivary cotinine was observed in cigarette smokers followed by hookah smokers. Salivary cotinine was negative in nonsmokers. The higher mean cotinine level in cigarette smokers compared to hookah smokers has been reported in several studies. However, due to global concerns regarding the use of hookah, level of cotinine in urine and other body fluids such as plasma, saliva and nasal secretions has been assessed with laboratory methods namely liquid chromatography GM, HPLC and ELISA (Table 3). Previous relevant studies have been mostly conducted in countries with a high prevalence of hookah smoking. High rate of hookah smoking among families has raised some concerns. In most cases cotinine levels were higher in cigarette smokers than hookah smokers. Macaron et al. found urinary cotinine levels to be higher in hookah smokers. However, this difference was not statistically significant (5.980 Tg/ml in cigarette smokers versus Tg/ml in hookah smokers) [4]. Bacha et al. reported salivary cotinine to be 87 ng/ml in cigarette smokers and 78 ng/ml in hookah smokers. They also found that the height and weight of smokers and size of waterpipe affected the level of cotinine [16]. Behera et al. found higher cotinine levels among cigarette smokers, although this difference was not significant [17]. However, in our study salivary cotinine level was significantly 270 Jan 2014; Vol. 25, No. 4 Rabiei et. al Comparison of Salivary Cotinine Concentration in Cigarette... Table 3: Salivary cotinine concentration in cigarette smokers versus waterpipe smokers Authors and Country Bacha et al 16 Method Salivary cotinine Cotinine in water pipe smokers 78 ng/ml Cotinine in cigarette smokers 87 ng/ml Shafagoj et al 2 Plasma cotinine-3 h post smoking ng/mL Not measured Shafagoj et al 4 Saliva-45 min postsmoking ng/mL Not measured Macaron et al 4 Urinary cotinine g/ml (6080 ng/ml) g/ml (5980 ng/ml) Behera et al 17 (India) Urinary cotinine g/ml (2379 ng/ml) g/ml (2739 ng/ml) Al Mutairi et al 6 (Kuwait) Urinary cotinine g/ml (678 ng/ml) g/ml (1321 ng/ml) Rabiei et al (Iran) Salivary cotinine ng/ml ng/ml higher among cigarette smokers compared to hookah smokers. On the other hand, results showed that one time hookah smoking per week increased cotinine levels more than twice the rate by smoking one cigarette (4.57 ng/ml increase by hookah compared to 1.84 ng/ml increase by cigarette smoking). It should be noted that we tried our best to match the conditions in order to eliminate the effect of confounding factors such as BMI, age, gender and size of water pipe. Shafagoj et al. reported that 3 hours after smoking serum cotinine level increased by ng/ml compared to thebaseline level before smoking. This rate was for salivary cotinine level [4]. Al-Muntari et al.stated that smoking 30 cigarettes is equivalent to 168 mg of nicotine while one time smoking ofmaassal is equivalent to 25 mg of nicotine [6]. In the study by Maritta S. Jaakkola et al, using a simple linear regression model, cotinine level increased by 5.5 ng/ml for each additional cigarette smoked by subjects smoking more than 20 cigarettes per 24 hours [18]. This value was 7.3 ng/ml in those smoking less than 20 cigarettes during 24 hours. This finding indicated that the rise in nicotine level is greater in those with less frequency of smoking. The habit of smoking, cigarette filter and more importantly cigarette brand may also affect the results. In our study, we used Winston Light with 0.6 mg nicotine per cigarette while in Shafagoj sstudy participants smoked regular cigarettes with twice the nicotine amount. Hookah smokingproduces20 times morepolycyclic aromatic hydrocarbons and 50 times more heavy polycyclic aromatic hydrocarbons compared toonecigarette. Level of produced carbon monoxide by water pipe is also 5 times higher. One study showed that 45 minutes of waterpipe smoking produced 40 times the smoke volume produced by cigarette smoking. Cigarette smoking averagely produces8-12 puffs yielding a volume of around 50 ml over a 5- to 7-minute period;whereas, waterpipe gives 50-2 puffsover minutes resulting in Lof smoke inhalation [4]. Monn and colleagues reported that particles produced by hookah smoke are ultrafine measuring micron attributed to the combustion of wood charcoal. Particles ranging in size from 5 to 10 Tm deposit primarily in the large airways. Particles that are 2-5 Tm deposit in the lower respiratory tract and T mparticles deposit in the terminal airways and alveoli [19]. Lung cancer, respiratory diseases and periodontal disease occur significantly more in relation to hookahsmoking. Jan 2014; Vol. 25, No The Journal of Islamic Dental Association of IRAN (JIDA) Winter 2014 ;25, (4) Rabiei et. al The correlation of cigarette smoking and oral cancer has been confirmed in many studies. Systematic reviews have reported increased prevalence of oral cancer as the result of hookah smoking but have not mentioned a definite correlation in this respect. Increasing the number of cigarettes smoked results in an increased risk of oral cancer while the number of hookah smoking sessions is usually low about 2-3 times per week [5]. Many people believe that filtration of smoke through water decreases the amount of nicotine. In contrast to this view, research demonstrates that only about 5% of the nicotine is dissolved in water. Moreover, waterpipe smokers may increase the duration of smoking and the volume of puffs to obtain the nicotine level necessary to reachthe pleasurable level of nicotine [4]. This study had some limitations. Participants had to quit smoking for 3 to 4 days prior to sampling to better find the difference in level of salivary cotininefollowing smoking one cigarette or one time hookah consumption. However, this was very difficult for participants. This study showed an increase in cotinine level as the result of hookah smoking; which totally contradicts the public misconception about hookah smoking even among families denouncing cigarette smoking. Reports show that hookah, traditionally popularin the Middle East, is making its way t

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